Role of Herpes Simplex Virus 1 γ34.5 in the Regulation of IRF3 Signaling

Abstract

During viral infection, pattern recognition receptors (PRRs) and their associated adaptors recruit TANK-binding kinase 1 (TBK1) to activate interferon regulatory factor 3 (IRF3), resulting in production of type I interferons (IFNs). ICP0 and ICP34.5 are among the proteins encoded by herpes simplex virus 1 (HSV-1) that modulate type I IFN signaling. We constructed a recombinant virus (ΔXX) that lacks amino acids 87 to 106, a portion of the previously described TBK1-binding domain of the γ34.5 gene (D. Verpooten, Y. Ma, S. Hou, Z. Yan, and B. He, J Biol Chem 284:1097-1105, 2009, https://doi.org/10.1074/JBC.M805905200). These 20 residues are outside the γ34.5 beclin1-binding domain (BBD) that interacts with beclin1 and regulates autophagy. Unexpectedly, ΔXX showed no deficit in replication in vivo in a variety of tissues and showed virulence comparable to that of wild-type and marker-rescued viruses following intracerebral infection. ΔXX was fully capable of mediating the dephosphorylation of eIF2α, and the virus was capable of controlling the phosphorylation of IRF3. In contrast, a null mutant in γ34.5 failed to control IRF3 phosphorylation due to an inability of the mutant to sustain expression of ICP0. Our data show that while γ34.5 regulates IRF3 phosphorylation, the TBK1-binding domain itself has no impact on IRF3 phosphorylation or on replication and pathogenesis in mice.IMPORTANCE Interferons (IFNs) are potent activators of a variety of host responses that serve to control virus infections. The Herpesviridae have evolved countermeasures to IFN responses. Herpes simplex virus 1 (HSV-1) encodes the multifunctional neurovirulence protein ICP34.5. In this study, we investigated the biological relevance of the interaction between ICP34.5 and TANK-binding kinase 1 (TBK1), an activator of IFN responses. Here, we establish that although ICP34.5 binds TBK1 under certain conditions through a TBK1-binding domain (TBD), there was no direct impact of the TBD on viral replication or virulence in mice. Furthermore, we showed that activation of IRF3, a substrate of TBK1, was independent of the TBD. Instead, we provided evidence that the ability of ICP34.5 to control IRF3 activation is through its ability to reverse translational shutoff and sustain the expression of other IFN inhibitors encoded by the virus. This work provides new insights into the immunomodulatory functions of ICP34.5.

Keywords: ICP0; ICP34.5; IRF3; RL1; TBK1; herpes simplex virus; innate immunity; interferons; recombinant viruses; viral pathogenesis.

FIG 1

Map of mutations; HSV-1 ICP34.5 fails to bind endogenous TBK1. (A) Schematic diagram of ICP34.5 variants. (B and C) Coimmunoprecipitation experiments showing interaction of ICP34.5 variants with endogenous beclin1 (B) or p32 and TBK1 (C). (D) Coimmunoprecipitation experiments comparing ICP34.5 strains F and 17 in binding TBK1. HEK-293T cells were transfected with hemagglutinin (HA)-tagged ICP34.5 variants. At 24 h posttransfection, the lysates were immunoprecipitated (IP) using anti-ICP34.5, anti-phospho-beclin1, or anti-HA antibody. The precipitates and lysates were analyzed by immunoblotting (IB) using anti-FLAG, anti-HA, anti-beclin1, anti-p32, anti-TBK1, or anti-ICP34.5 antibody. The immunoblots are representative of the results of at least three independent experiments.

FIG 2

ICP34.5 influences phosphorylation and nuclear translocation of IRF3, but not through amino acids 68 to 106. (A) Immunofluorescence image showing staining for viral marker VP16 (red), IRF3 (green), and nuclear marker (blue; DAPI [4′,6-diamidino-2-phenylindole]). HFFs were treated with 10 IU/ml IFN-β for 18 h prior to infection. The cells were infected at an MOI of 5 and fixed 10 h postinfection. (B) Quantification of IRF3 nuclear translocation by the percentage of cells that exhibited predominant nuclear IRF3 staining with a minimum of 2,000 cells/group/replicate. Image quantification was performed using the ImageJ distribution, Fiji. The error bars represent standard errors of the mean (SEM). Statistical significance was determined by two-way analysis of variance (ANOVA). **, P < 0.01. (C) Lysates were analyzed by immunoblotting using anti-phospho-IRF3, anti-IRF3, or anti-β-actin antibody. HFFs were infected at an MOI of 5 and harvested 10 h postinfection. The immunoblots are representative of the results of three independent experiments.

FIG 3

Amino acids 87 to 106 of ICP34.5 do not impact viral replication or virulence. (A) Multiple-step growth curves showing replication of Δ34.5, ΔXX, or the marker rescue control ΔXX-R in primary HFFs. The HFFs were treated with vehicle control or 10 IU/ml IFN-β for 18 h and then infected at an MOI of 0.1 with recombinant virus. The data points represent means ± SEM and are a summation of the results of 3 independent experiments. (B) Viral titers in tear film, trigeminal ganglion, periocular skin, and brain following bilateral corneal infection with 2 × 10⁶ PFU of virus per eye of C57BL/6 mice. The dashed lines represent the limit of detection (LOD). The data are a summation of the results of two independent experiments. (C) Survival of C57BL/6 mice following i.c. infection. The mice were infected with 100 PFU of Δ34.5 (n = 5), ΔBBD (n = 9), ΔXX (n = 10), or strain 17 (n = 18) and monitored for attainment of endpoint survival criteria. The data are a summation of the results of 4 independent experiments. Statistical significance was determined by two-way ANOVA (A and B) and log rank test (C). ***, P < 0.001; ns, not significant. dpi, days postinfection.

FIG 4

The BBD (amino acids 68 to 87) is necessary to bind TBK1 under overexpression conditions. The Western blot of coimmunoprecipitation experiments shows interaction of ICP34.5 variants with overexpressed TBK1. HEK-293T cells were cotransfected with HA-tagged ICP34.5 variants and TBK1 or vector control. At 24 h posttransfection, the lysates were immunoprecipitated using an anti-ICP34.5 antibody. The precipitates and lysates were analyzed by immunoblotting using anti-TBK1 or anti-HA antibody. The immunoblots are representative of the results of three independent coimmunoprecipitation experiments.

FIG 5

ICP34.5 is required for the maintenance of ICP0 expression. (A) Western blot showing expression of phosphorylated eIF2α during infection. HFFs were infected at an MOI of 2.5 and harvested 10 h postinfection. Samples were analyzed by immunoblotting using anti-phospho-eIF2α and anti-β-actin antibody. (B) Quantification of MFI per cell of IF images of various viral proteins during infection with Δ34.5 or strain 17. HFFs were infected at an MOI of 2.5 and imaged 8 h postinfection. (C) Western blot showing expression of viral proteins in HSV-1-infected HFFs. The cells were harvested 10 h postinfection and analyzed by immunoblotting for various viral proteins. Lysates were loaded at 1 × 10⁶ (1×) or 2 × 10⁵ (0.2×) cells per well. Quantification was performed on the 1× bands relative to β-actin. (D) Sample images used to quantify the fluorescence intensity of viral proteins in Fig. 4B, E, and F. The warmer colors represent greater ICP0 fluorescence in the heat-mapped images. (E and F) Quantification of ICP0 by MFI via IF microscopy of HSV-1-infected HFFs (E) and primary TG neurons (F). The HFFs were infected at an MOI of 2.5 and imaged at the indicated times. The primary TG neurons were infected at the indicated MOI and imaged 10 h postinfection. (G) Immunoblot of HFFs infected with various mutant HSV strains at an MOI of 2.5 and harvested 10 hpi. All the Western blots are representative of the results of at least 3 experiments. For all image quantifications, each n represents an average of at least 2,000 cells with 6 per group. The bars represent means and SEM over at least 3 independent experiments. Image quantification was performed using the ImageJ distribution, Fiji. Units for numbers on the left sides of panels A, C, and G are kilodaltons. Statistical significance was determined by two-way ANOVA. *, P < 0.05.